The overall goal of this research is to elucidate the mechanisms involved in the regulation and function of muscarinic acetylcholine receptors (mAChR) in the mammalian heart. Activation of the mAChR causes a decrease in the rate and force of contraction, and regulates the activities of adenylyl cyclase, phospholipase C, and potassium and calcium channels. While most cardiac mAChR are of the m2 subtype, a wide variety of pharmacological, physiological, immunological, biochemical, and molecular biological evidence indicates that the m1 receptor is also present at very low levels in the heart. The function and number of mAChR can be decreased by continued exposure of agonist. The PI has recently demonstrated that the beta-adrenergic receptor kinase and beta-arrestin synergistically regulate signal transduction by the m2 receptor in transfected cells. This proposal will test the hypothesis that the m2 and m4 receptors exhibit different selectivities for regulation by subtypes of G-protein receptor kinases and arrestins, and determine the functional consequences of elimination of the phosphorylation sites on mAChR function and regulation. Short-term agonist exposure also causes sequestration of receptors from the cell surface. Previous results indicate that the m1 and m2 receptors use distinct cellular mechanisms for agonist-induced internalization. This proposal will test the hypothesis that a portion of the m2 receptor in the sixth transmembrane domain and the third cytoplasmic domain (i3)form an contiguous sequestration domain. This proposal will identify specific residues and determine the effects of lack of sequestration on the regulation of muscarinic responsiveness. This proposal will also alter expression and introduce mutations into mAChR in the heart in vivo to provide unique information on the regulation of mAChR signaling in the heart. This proposal will use m1 knockout mice generated by this laboratory to determine the role of the m1 receptor in the physiological actions of acetylcholine and the regulation of mAChR expression in the heart. This project will determine the effect of disruption of the m2 receptor on cardiac function, responses to ACh, and expression of other mAChR subtypes, and will introduce mutations into the m2 receptor in mice to test the role of phosphorylation and sequestration on receptor function and signaling in the heart in vivo. This research will provide valuable new information on the basic mechanisms regulating the expression and function of mAChR in the heart. In addition, this research may aid in understanding the etiology of a variety of cardiac abnormalities.